Patients with malignant gliomas have a life expectancy of only 15 months due to rapid recurrence of the tumor after treatment. A large effort is therefore ongoing to characterize and target the cancer-initiating cells that cause recurrence of these cancers. Viruses that replicate selectively in tumor cells and spread their progeny throughout the tumor [oncolytic viruses (OVs)] present a strong potential for efficient treatment of malignant gliomas. However, results from clinical trials did not provide the expected results, thus highlighting the importance of identifying the factors that limit the efficacy of OVs in the clinics. We have observed that even though herpes simplex virus (HSV)-derived OVs efficiently kill primary glioma cells their replication is compromised in the cancer-initiating cells derived from the same tumor. Thus, we hypothesize that OV resistance of tumor-initiating cells is partially responsible for the clinical inefficacy of these viruses. To improve the efficacy of this treatment we plan to identify the mechanisms that induce OV resistance in glioma-initiating cells. Micro-RNAs (miRNAs) are non-coding RNA molecules that regulate expression of target genes by silencing specific mRNAs and are critical in controlling cellular pathophysiology. Their expression is altered during tumorigenesis and cancer treatment. Mammalian miRNAs were also found to be altered upon infection of viruses and to regulate their tropism. Because miRNAs can simultaneously inhibit translation of different mRNAs that belong to the same ontologic pathway, we believe that analysis of miRNA expression following OV infection is an efficient strategy to identify the mechanisms that induce OV resistance in glioma initiating cells. Moreover, because it is easy to engineer OVs with sequences that alter the activity of specific miRNAs, we think that this analysis will provide the tools to engineer more efficient OVs for cancer therapeutics. Our goal is to identify a miRNA signature of OV resistance in glioma initiating cells and to test if viral treatment can be enhanced by altering this signature. To accomplish this goal we will first use a human miRNA RT-PCR array to analyze miRNA expression changes following OV infection of cancer initiating cells and of established primary cancer cells derived from GBMs resected in our department (Aim 1). Then, we will attempt to increase OVs anti-cancer efficacy in vitro and in vivo by altering the expression of the miRNAs identified in aim 1 (Aim 2). This will be initially performed by analyzing OVs efficacy in vitro after transient transfection of glioma cells with miRNAs inhibitors or amplifiers, and then by engineering OVs with the selected miRNA targeting sequences and testing them in vivo. These studies are therefore designed to characterize brain tumor initiating cells in response to OV treatment and to identify new targets for therapeutic intervention. If successful, they will provide new means to generate OVs that exhibit enhanced efficacy in clinical applications. PUBLIC HEALTH RELEVANCE: With this project we plan to identify the mechanisms that induce resistance to viral therapy in cancer cells. For this purpose we will analyze the presence of newly identified molecules that have a very powerful capacity to regulate the mechanisms responsible of tumor biogenesis and viral tropism. We will thus test if these molecules interfere with virus killing of tumor cells and then build new viruses that can target these molecules. We expect that such viruses will have a stronger therapeutic potential.